Identification of biomarkers of shrinkage modes after neoadjuvant therapy in HER-2 positive breast cancer

Abstract

Purpose: A nomogram to predict shrinkage modes after neoadjuvant therapy (NAT) was constructed in HER-2 positive (HER2+) breast cancer. The value and mechanism of targeting long noncoding RNA (lncRNA) as efficacy prediction biomarker was also evaluated.

Methods: All enrolled patients received six cycles of chemotherapy (Docetaxel + Carboplatin) and anti-HER-2 dual-targeted therapy (Trastuzumab + Pertuzumab) before surgery. According to pathological three-dimensional (3D) models of residual tumor from 71 HER2+ patients, shrinkage modes were divided into concentric shrinkage mode (CSM) and non-CSM (NCSM). LncRNAs in core biopsy tissues in the CSM and NCSM groups were selected by microarray and validated by RT-PCR. A nomogram was constructed to predict shrinkage modes after NAT in combination with clinical-pathological and transcriptome signatures. Cell proliferation was used CCK-8 and colony formation assay. PAPIS Kit was used to perform nuclear and cytoplasmic separation. The cell drug resistance assays were to explore the value of paclitaxel. The ChIRP-MS assay was to search RNA-binding proteins and verified by WB. Cell cycle analysis was carried out by flow cytometry.

Results: Independent predictors of NCSM were lymph nodes downstaging after NAT, mammographic malignant calcification, hormone receptor expression, and RUVBL1-AS1 expression. A nomogram was constructed in combination with these predictors, which showed an area under the curve of 0.883, supporting the predictive power of the method. Overexpression of RUVBL1-AS1 inhibited HER2+ cells proliferation. Overexpression of RUVBL1-AS1 increased the number of cells in G1/S phase and decreased that of cells in G2 phase. RUVBL1-AS1 increased paclitaxel resistance and downregulated VCP expression. RUVBL1-AS1 affects cell cycle progression by downregulating VCP, resulting in the reduction of cells in G2/M phase, thereby weakening the sensitivity to paclitaxel.

Conclusion: The nomogram could accurately predict shrinkage modes after NAT, and may help guide the individualized selection of breast conserving surgery candidates after NAT. RUVBL1-AS1 might be a promising therapeutic target of paclitaxel-based chemotherapy inHER2+ breast cancer.

Keywords: RUVBL1-AS1; breast cancer; neoadjuvant therapy; nomogram; shrinkage modes.

Figure 1.

The shrinkage modes of residual tumors after NAT ^[10^]. (A) The shrinkage modes. The surgical pCR and solitary lesion without surrounding lesions were divided into CSM. Other shrinkage modes were divided into NCSM. (B) The specimen was cut into several blocks at 5-mm intervals based on the markers. (C) The extent of residual tumors was delineated under microscope. (D) The pathology 3D reconstruction model of residual tumor after NAT. (E) The consort diagram of the study. (F) The different expression and AUC of RUVBL1-AS1 in predicting NCSM after NAT. (G) The nomogram of predicting NCSM after NAT. (H) The ROC curve of the nomogram.

Figure 2.

RUVBL1-AS1 inhibits breast cancer cell proliferation. (A) RUVBL1-AS1 was low expression in tumor tissues. (B) TCGA database showed the same result. (C) The expression level of RUVBL1-AS1 in different cells. (D) The overexpression of RUVBL1-AS1 inhibited the growth of HER2+ cells. (E) Representative images of the cloning formation assay showed that overexpression of RUVBL1-AS1 inhibited the growth of HER2+ cells. (F) The growth rate of xenograft was slower, and the weight and volume decreased in P-RUVBL1-AS1 compared with the control.

Figure 3.

The exploration of potential mechanism of RUVBL1-AS1. (A) Knockdown of RUVBL1-AS1 significantly decreased the proportion of cells in G1/S phases and increased G2/M phases. (B) Overexpression of RUVBL1-AS1 significantly increased the proportion of cells in G1/S phases and decreased G2/M phases. (C) The nuclear-cytoplasmic fractionation assay of RUVBL1-AS1. (D) Schematic of the ChIRP-MS assays. (E) Knockdown of RUVBL1-AS1 could up-regulate the mRNA levels of VCP in HER2+ cells, while overexpression of RUVBL1-AS1 showed the opposite effect. (F) WB analyses showed that knockdown of RUVBL1-AS1 increases the protein levels of VCP, while overexpression of RUVBL1-AS1 showed the opposite effect. (G) The mechanism diagram of this study.